Potential therapeutic treatment using stem cells requires elucidation of mechanisms that control stem cell renewal and differentiation. Manipulation techniques to induce stem cell differentiation into tissue-specific lineages will have to be developed and tested. Stem cell-based technologies for tissue engineering can then be envisioned. However, achievement of such goals would first require the knowledge and application of engineering principles to integrate biological and physical signals and amplify the molecular signaling mechanism(s) in order to promote and enhance selective stem cell proliferation and differentiation. We have recently demonstrated for the first time that use of non-invasive electrical stimulus can be applied to manipulate mesenchymal stem cell (MSC) differentiation. Although electrical stimulus has been used beneficially in the past to induce diverse cellular and molecular responses, neither the use of electrical stimulus has been optimized nor have the electrocoupling mechanisms regulating human MSC differentiation have been explored. We therefore propose to combine unique, novel physical and optical techniques to (1) optimize the electrical stimulus parameters for facilitated hMSC osteogenic differentiation and (2) elucidate the role of integrin-mediated signaling pathways, including the mitogen-activated protein kinase signaling mechanisms. We propose to use quantum dot-conjugated integrins to determine changes in the integrin dynamics on the human MSC surface and correlate them with integrin down-regulation at the different stages of osteogenic differentiation. Hypotheses and at least 2 alternate electrocoupling mechanisms are proposed based on integrin redistribution and clustering in response to electrical stimulus. Optimal application of electrical stimulus and elucidation of electrocoupling mechanisms will lay the foundation for a novel biotechnology approach to manipulate stem cell differentiation, paving the way to establish a new paradigm for tissue engineering methodologies that integrates physical and molecular techniques. The long- term research objectives would include manipulation and control of stem cell proliferation and differentiation by the optimal use of physical stimuli and, thereby, regulate the integrity and functionality of stem cell-derived engineered tissue constructs. [unreadable] [unreadable] [unreadable]